ABSTRACT The main goal of this project is to dissect gene regulatory functions of Tet enzymes in ESCs, and define the significance of noncanonical (non-enzymatic) roles of these proteins in gene repression and in stem cell biology. The Ten-eleven-translocation (Tet) enzymes (Tet1, Tet2, Tet3) regulate gene expression program in ESCs and during development. They facilitate DNA demethylation by oxidizing 5-methylcytosine (5mC) to 5- hydroxymethylcytosine and other derivatives. The enzymatic function of Tet enzymes in DNA demethylation and its significance in ESCs and development has been well investigated as part of my prior work in the field. However, recent findings from my lab strongly suggest that key biological roles of Tet proteins are independent of their catalytic activity. We have found that more genes are differentially expressed in Tet1 knockout ESCs than in Tet1 catalytic mutant ESCs (vs. wild type ESCs). Majority of these genes are overexpressed, suggesting that noncatalytic functions of Tet enzymes regulate gene repression in ESCs and contribute to their biology. Consistently, we found that catalytically inactive Tet enzymes rescued some of the phenotypes of Tet deficient ESCs. These observations build the initial premise for this project. We hypothesize that in addition to their DNA demethylase activity, Tet enzymes regulate the epigenome independent of their enzymatic activity. This involves targeting gene regulatory elements to recruit chromatin repressive complexes for proper gene repression. This constitutes a novel layer of epigenetic regulation that influences key features of ESC biology including proliferation, pluripotency and lineage specification. Using a recently developed platform in my lab that consists of Tet catalytic mutant and knockout ESCs, we will apply molecular, cellular and developmental approaches to dissect the catalytic dependent and independent requirements of Tet enzymes in stem cells, and establish the significance of noncanonical roles of Tet enzymes in ESC biology. We propose the following three aims: (1) we will define gene regulatory elements targeted by noncanonical functions of Tet enzymes and map the noncatalytic Tet domains required for their regulation. (2) We will establish the noncanonical roles of Tet enzymes in recruiting chromatin repressive complexes to gene regulatory elements for gene silencing. (3) We will define the biological relevance of Tet-mediated noncanonical gene repression in ESC proliferation, pluripotency and endodermal differentiation. Our findings will reveal novel epigenetic mechanisms of regulation of ESCs by Tet enzymes. This will enhance our understanding of the epigenetic networks in stem cells and will open future opportunities for investigating Tet-mediated gene regulation during development and in diseases.